Purification and Characterization of the Soluble F1-ATPase of Oat Root Mitochondria

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Abstract
The properties of the soluble moiety (F1) of the mitochondrial H+-ATPase from oat roots were examined and compared to those of the native mitochondrial membrane-bound enzyme. The chloroform soluble preparation was purified by Sephadex G-200 and DEAE-cellulose chromatography. The purified F1 preparation contained major polypeptides corresponding to α, β, γ, δ, and ε of apparent molecular mass 58, 55, 35, 22, and 14 kilodaltons, respectively. The purified F1-ATPase, like the native enzyme, was inhibited by azide (I50 = 10 micromolar), nitrate (I50 = 7-10 millimolar), 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (I50 = 1-3 micromolar), and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (I50 = 3 micromolar). F1-ATPase activity was stimulated by bicarbonate but not by chloride. In both the native and the F1-form of the ATPase, ATP was hydrolyzed in preference to GTP. The results indicate that these properties of the native membrane-bound mitochondrial ATPase have been conserved in the purified F1. In contrast to the membrane-bound enzyme, the F1-ATPase was not inhibited by oligomycin or by N,N′-dicyclohexylcarbodiimide. The mitochondrial F1-ATPase from oat roots is analogous to other known F1F0-ATPases.